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The antibacterial activity of Cinnamaldehyde against Methicillin-resistant Staphylococcus aureus in Bovine Mastitis is investigated in this study, providing insights into inhibition mechanisms and elucidating the effects on bacterial cell membranes. The Minimum Inhibitory Concentration and Minimum Bactericidal Concentration were determined in this study. In addition, growth curves and a time-kill assay were constructed to assess the antibacterial activity of Cinnamaldehyde. The study revealed that the MIC values ranged from 62.5 to 125 μg/mL, and the MBC values ranged from 125 to 250 μg/mL. The presence of sublethal concentrations of Cinnamaldehyde impeded bacterial growth, while high concentrations demonstrated a significant and rapid bactericidal effect. Subsequently, we examined cell morphology using SEM and TEM, evaluated membrane integrity via laser confocal fluorescence microscopy, and measured levels of β-galactosidase, extracellular DNA release, LDH activity, and ROS, to assess the antibacterial mechanism of Cinnamaldehyde. The findings indicated that with higher concentrations of Cinnamaldehyde, Methicillin-resistant Staphylococcus aureus demonstrated significant morphological alterations and disruption of both the cell wall and membrane. Furthermore, Cinnamaldehyde disrupted the integrity of membranes and increased permeability of the outer membrane in a manner dependent on its concentration. Cinnamaldehyde notably triggered the release of β-galactosidase, extracellular DNA, and LDH, in addition to elevating cellular ROS levels. Finally, the effect of Cinnamaldehyde on the transcription levels of genes related to cell membrane synthesis was assessed using RT-qPCR, and the effect of Cinnamaldehyde on the total protein content of Methicillin-resistant Staphylococcus aureus cells was assessed using WB. The RT-qPCR results showed that Cinnamaldehyde at 1xMIC notably upregulated the transcription levels of genes related to fatty acid biosynthesis in Methicillin-resistant Staphylococcus aureus cell membranes, with a significant or highly significant effect. The WB results showed that Cinnamaldehyde exerts its antibacterial action by suppressing protein expression in Methicillin-resistant Staphylococcus aureus. These findings illustrate that Cinnamaldehyde exerts a potent inhibitory effect on Methicillin-resistant Staphylococcus aureus, establishing a fundamental foundation for the potential use of Cinnamaldehyde essential oil as an antibacterial agent in the treatment of bovine mastitis, in accordance with established scientific standards. [ABSTRACT FROM AUTHOR]